摘要Background:Alfalfa hay and corn stover are different type of forages which can significantly impact a cow's lactation performance,but the underlying metabolic mechanism has been poorly studied.We used biomarker and pathway analyses to characterize related biomarkers and pathways based on urine metabolomics data from different forage treatments.Urine was collected from 16 multiparous Holstein dairy cows fed alfalfa hay (AH,high-quality forage,n =8) and corn stover (CS,low-quality forage,n =8) respectively.Gas chromatography-time of flight/mass spectrometry (GC-TOF/MS) was performed to identify metabolites in urine and the metaboanalyst online platform was used to do biomarker and pathway analysis.Results:Hippuric acid (HUA) and N-methyl-glutamic (NML-Glu) indicated the most significant difference between the two diets,when statistically validated by biomarker analysis.HUA was also validated by standard compound quantitative method and showed significant higher concentration in CS group than AH group (2.8282 vs.0.0005 mg/mL;P ＜ 0.01).The significant negative correlation between milk yield and HJA (R2=0.459;P ＜ 0.01) and significant positive correlation between milk yield and NML-Glu (R2 =0.652;P ＜ 0.01) were characterized.The pathway analysis revealed that these different metabolites were involved in 17 pathways including 7 influential pathways (pathway impact value ＞ 0):Tyr metabolism,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,galactose metabolism,Phe,Tyr and Try biosynthesis,purine metabolism,and glycerolipid metabolism.Based on the metabolome view map,the Phe,Tyr and Try biosynthesis pathway exhibited the highest impact value (0.50),and the Holm-Bonferroni multiple testing-based analysis revealed the most significant difference in the Tyr metabolism pathway (Holm P =0.048).Conclusions:The identified HUA and NML-Glu may serve as potential biomarkers for discriminating CS and AH diets and could be used as candidates for milk yield related mechanistic investigations.Integrated network pathways associated with related metabolites provide a helpful perspective for discovering the effectiveness of forage quality in lactation performance and provides novel insights into developing strategies for better utilization of CS and other low-quality forage in China.